ReviewOligomerization of opioid receptors: generation of novel signaling units
Introduction
Oligomerization of G-protein-coupled receptors (GPCRs) is a burgeoning field of research that is significantly advancing and remodeling our understanding of GPCR regulation and function 1••., 2.. Crucial to this progress is the characterization of opioid receptor homo-oligomerization and hetero-oligomerization and this is vital to a detailed understanding of the endogenous opioid system. This review documents the features of opioid receptor homo-oligomerization and hetero-oligomerization. At each stage, the consequences and implications of opioid receptor–receptor interactions are addressed, such as drug design, in vivo opioid system regulation and function and the relevance to development of analgesic tolerance. Additionally, the specificity of opioid receptor oligomer formation is discussed, particularly in relation to hetero-oligomerization with other GPCRs.
Section snippets
Opioid receptor homo-oligomers
The homo-oligomeric association of opioid receptors has been documented by numerous reports. Using Western blot analysis of heterologous cell expression systems, we and others have demonstrated immunoreactive bands corresponding to monomers, dimers, and higher-order oligomers for the μ [3••], δ 3••., 4., 5•., and κ [4] opioid receptors (MOR, DOR and KOR). In addition, MOR antibodies labeled receptor complexes in mouse brain corresponding to receptor dimers [6], suggesting that oligomerization
DOR homo-oligomerization
The properties of the DOR homo-oligomer have been most extensively examined. Differentially epitope-tagged DOR constructs coimmunoprecipitated when expressed in COS, CHO, or HEK293 cells 7., 8••. and DOR oligomers were detected in HEK293 cells using bioluminescence resonance energy transfer (BRET) and time-resolved fluorescence resonance energy transfer (FRET) assays [8••]. BRET and time-resolved FRET are relatively new approaches to examining GPCR oligomerization that rely on energy transfer
Dynamic regulation?
The existence of dynamic regulation of DOR oligomerization is a contentious issue. Using Western blots, cross-linker mediated dimerization of the DOR was reported to be attenuated by agonists [7]. A dose-dependent relative increase in monomers was observed with DOR-selective and non-selective agonists (DADLE, DSLET, DPDPE, etorphine; Box 1), but not following treatment with MOR-selective agonists (morphine, DAMGO) or non-selective antagonists (naloxone) [7]. In contrast, using both BRET and
Structure
A general structural basis for GPCR oligomerization has yet to be established. It is commonly thought that multiple factors, such as transmembrane domain hydrophobicity, extracellular disulfide bonds and intracellular coiled-coil interactions, cooperatively mediate oligomerization [1••]. Other factors could additionally account for receptor–receptor interactions, such as cross-linking through accessory proteins or leucine zipper interactions. Sensitivity to reducing agents implicate disulfide
Physiological role
Homo-oligomerization of opioid receptors raises questions regarding its physiological role. Oligomerization may be necessary for signal amplification, such that agonist activation of a limited number of receptors triggers activation of other associated receptors, resulting in increased effector coupling, receptor phosphorylation, G-protein uncoupling and internalization of an entire receptor complex. Interestingly, a role for oligomerization in trafficking of the dopamine D2 receptor to the
Hetero-oligomerization between opioid receptors
Overall, the three opioid receptors share approximately 60–65% amino acid identity, with the transmembrane domains being among the most highly conserved regions (Fig. 1). Given that homo-oligomerization had been demonstrated and given the expectation that transmembrane interactions exist in these complexes, hetero-oligomerization between opioid receptors was hypothesized.
DOR–KOR hetero-oligomers
Heterodimerization between DOR and KOR was the first opioid receptor complex identified [4]. Differentially epitope-tagged DOR and KOR expressed in HEK293 or COS cells coimmunoprecipitated and this receptor complex displayed many novel characteristics [4]. The DOR–KOR heterodimer had an altered ligand-binding profile, such that KOR-selective agonists (U69593, dynorphin A) and antagonists (norbinaltorphimine) and DOR-selective agonists (DPDPE) and antagonists (TIPPΨ, BNTX; Box 1) had decreased
MOR–DOR hetero-oligomers
Interaction between MOR and DOR receptors has long been postulated and is extensively documented [15]. We recently demonstrated direct interaction between these two receptors. Differentially epitope-tagged MOR and DOR also coimmunoprecipitated from COS cells and this hetero-oligomeric complex also displayed a host of unique features [3••]. A novel binding site was revealed by an altered rank order of potency: selective synthetic agonists (DADLE, DPDPE, DAMGO, morphine) had a reduced affinity
MOR–KOR interaction?
The remaining possible hetero-oligomeric complex that could be formed between opioid receptors would be as a result of MOR–KOR interaction. Coexpression of these receptors did not result in coimmunoprecipitation [4]; however, cell-surface expression of the two receptors was not confirmed. If MOR and KOR do not hetero-oligomerize, the opposing physiology of the two systems may provide a rationale. The KOR generally antagonizes MOR-mediated actions such as analgesia, tolerance, reward, learning
Hetero-oligomerization between opioid receptors and other GPCRs
If different opioid receptors hetero-oligomerize, do they also directly interact with other GPCRs? Recent studies show that DOR and KOR are capable of interacting with the β2-adrenoceptor 8••., 27., however the significance of this interaction remains uncertain. Coimmunoprecipitation of the β2-adrenoceptor with the KOR [27], and the DOR 8••., 27. was demonstrated in heterologous cell systems. To address concerns that this interaction may be an artifact, the interaction of these two receptors
Opioid receptor oligomerization: questions, implications and opportunities
The organization of opioid receptors into oligomeric complexes raises many intriguing questions. For example, what is the functional receptor unit and do monomers, dimers and higher order oligomers coexist on the cell surface? Multiple studies have demonstrated opioid receptor species corresponding to all of these receptor states, but how accurately does Western blot analysis reflect cell-surface expression? Interestingly, MOR and DOR homo-oligomers were observed following coimmunoprecipitation
Conclusions
Oligomerization adds a previously unappreciated level of complexity to opioid receptor function and is a mechanism by which the products of a limited number of receptor genes may give rise to a greater diversity of signaling units with unique properties (Fig. 2). New approaches to therapeutic drug design and discovery directly arise from the identification of opioid receptor hetero-oligomers. Numerous questions surrounding opioid receptor homo-oligomerization and hetero-oligomerization remain,
Update
Chronic morphine treatment was recently demonstrated to induce recruitment of the DOR from intracellular stores to the plasma membrane in both cultured cortical neurons and rat spinal cord [36]. DOR upregulation was solely mediated through morphine activation of coexpressed MORs [36], suggesting a possible role for MOR–DOR hetero-oligomerization in DOR trafficking.
Acknowledgements
BF O'Dowd and SR George receive support from the Canadian Institutes of Health Research and the National Institute on Drug Abuse. The authors thank Samuel PLee for his graphical design expertise.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
References (36)
- et al.
Oligomerization of μ- and δ-opioid receptors. Generation of novel functional properties
J Biol Chem
(2000) - et al.
Dimerization of the δ opioid receptor: implication for a role in receptor internalization
J Biol Chem
(1997) - et al.
Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer
J Biol Chem
(2001) - et al.
Increased biological activity of dimers of oxymorphone and enkephalin: possible role of receptor crosslinking
Biochem Biophys Res Commun
(1982) - et al.
Analysis of [3H]bremazocine binding in single and combinatorial opioid receptor knockout mice
Eur J Pharmacol
(2001) - et al.
δ-opioid receptor subtypes and cross-talk with μ-receptors
Trends Pharmacol Sci
(1993) - et al.
Opioids and their complicated receptor complexes
Neuropsychopharmacology
(2000) - et al.
δ-Opioid receptor agonists produce antinociception and [35S]GTPγS binding in μ receptor knockout mice
Eur J Pharmacol
(2000) - et al.
Retention of supraspinal delta-like analgesia and loss of morphine tolerance in δ opioid receptor knockout mice
Neuron
(1999) - et al.
Quantitative autoradiographic mapping of μ-, δ- and κ-opioid receptors in knockout mice lacking the μ-opioid receptor gene
Brain Res
(1997)
Opposing actions of the μ-opioid receptor
Trends Pharmacol Sci
Insights into mu opioid pharmacology The role of mu opioid receptor subtypes
Life Sci
Opioids: first lessons from knockout mice
Trends Pharmacol Sci
Multiple opiate receptors on neurons of the mammalian central nervous system. In vivo and in vitro studies
Life Sci
Dual ultrastructural immunocytochemical labeling of μ and δ opioid receptors in the superficial layers of the rat cervical spinal cord
Brain Res
Oligomerization of G-protein-coupled transmitter receptors
Nat Rev Neurosci
G protein coupled receptor dimerization: implications in modulating receptor function
J Mol Med
G-protein-coupled receptor heterodimerization modulates receptor function
Nature
Cited by (125)
Interaction of δ and κ opioid receptors with adenosine A<inf>1</inf> receptors mediates cardioprotection by remote ischemic preconditioning
2013, Journal of Molecular and Cellular CardiologyCitation Excerpt :Peart and Gross did pioneering work demonstrating in a rat model of coronary artery occlusion that protection of myocardium against ischemic/reperfusion injury by δ opioid receptor activation requires adenosine A1 receptor activity, and vice versa, indicating that these receptors must interact in mediating cardioprotection [22]. Influenced by the increasing body of knowledge of G-protein coupled receptor interactions, specifically the possibility of hetero-oligomerization of these receptors [22–26], we have focused in this study on opioid–adenosine receptor interactions, demonstrating for the first time, in isolated rabbit cardiomyocytes, that limb ischemia/reperfusion based rIPC cardioprotection requires functional interaction of the δ opioid receptor and the κ opioid receptor with the adenosine A1 receptor. In these studies, all experiments in animals conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011).
Quaternary structure predictions and structural communication features of GPCR dimers
2013, Progress in Molecular Biology and Translational ScienceCitation Excerpt :Dimerization/oligomerization may be a strategy to diversify and extend the signaling properties that are intrinsic in each individual receptor gene (reviewed also in Refs. 110–112). ORs have been extensively studied in this regard.113–115 It was shown that coexpressed δ- and κ-ORs can generate a different binding pattern and synergistic effects on MAP-kinase.116
G-protein-coupled heteromers: Regulation in disease
2013, Methods in EnzymologyInteractions between NMDA and dopamine receptors: A potential therapeutic target
2012, Brain ResearchCitation Excerpt :More importantly, in both animal and human studies, there has been considerable evidence demonstrating the important role of receptor–receptor interactions in neuropsychiatric diseases, which exhibit dysfunction in multiple neurotransmitter systems. Given the oligomeric nature of dopamine and NMDA receptors, it might be feasible to develop bivalent drugs for receptor heteromers generated by linking two monovalent drugs (Levac et al., 2002), or to modulate direct receptor–receptor interactions pharmacologically (Pei et al., 2010). Instead of simply blocking or activating one dysfunctional receptor system, development of pharmacological agents selective for regulating both dopamine and NMDA receptors simultaneously could be the next advance in treating neuropsychiatric disease.